Porcine erysipelas is a swine disease caused by infection with Erysipelothrix rhusiopathiae wherein infected swine suffers from symptoms such as sepsis in acute cases, hives in subacute cases, or endocarditis and arthritis in chronic cases. Around 3,000 swine per year have been reported to have the disease which is a great deal of damage to a stockbreeder. Erysipelothrix rhusiopathiae is pathogenic either to food animals such as wild boar, whales, chickens and turkeys in addition to swine and is specified as one of supervisory infectious diseases in the Protective Act of Livestock Diseases. Porcine erysipelas is also zoonosis that causes erysipeloid in human and is of importance in view of meat hygiene. There are a number of serotypes in Erysipelothrix rhusiopathiae, among which serotypes 1 and 2 cause most of porcine erysipelas in swine.
For protection of porcine erysipelas infections, there have hitherto been used attenuated live vaccines, i.e. freeze-dried live vaccines prepared by using Koganei strain which is an attenuated strain of Erysipelothrix rhusiopathiae prepared by subculturing virulent Erysipelothrix rhusiopathiae in a medium supplemented with acriflavine for a long period; inactivated vaccines, i.e. bacteria vaccines prepared by treating a culture of virulent Erysipelothrix rhusiopathiae with formalin and rendering the whole cells and extracellular products be adsorbed to aluminum hydroxide gel; and component vaccines, i.e. ones comprising a fraction of non-purified surface proteins of the cells extracted from the whole cells with an aqueous alkali solution. Attenuated live vaccines are thought to be much less costly since they may be efficacious with only one administration in a small amount. However, it is indicated they are also problematic in that they are pathogenic in mice to induce arthritis, that they exhibit severe side effects in swine with a low antibody level or SPF swine, and that the vaccine strain is isolated from the lesion of swine suffering from porcine erysipelas.
As a new type of vaccines, research and development is on-going for recombinant vaccines by the use of genetic recombination technique. Galan and Timony immunized mice with a lysate of E. coli transfected with a recombinant phage expressing genes from a part of Erysipelothrix rhusiopathiae genome and performed a challenge test with Erysipelothrix rhusiopathiae to observe that 14 to 17% of the immunized mice escaped from death after infection. Furthermore, they revealed that the proteins encoded by the genes are ones having molecular weight 66, 64, and 43 kDa from their reactivity with an immune serum against the lysate and demonstrated that these proteins could be protective antigens to Erysipelothrix rhusiopathiae infection (see e.g. Non-patent reference 1).
Makino et al. expressed a gene coding for a surface protein of a molecular weight 64 kDa (named “SpaA”) from type 2 Erysipelothrix rhusiopathiae Tama 96 strain in E. coli, immunized mice with live cells of the resulting recombinant E. coli, and performed a challenge test with Erysipelothrix rhusiopathiae to demonstrate that SpaA protein had protective activity to infection. They also revealed that SpaA protein had a sequence of 606 amino acid residues wherein a signal peptide consisting of 29 amino acids is at its N-terminal and eight homologous sequences of repeat, each repeat consisting of 20 amino acids excepting the 8th repeat which consists of 19 amino acids, are at its C-terminal (see e.g. Non-patent reference 2).
Imada et al. investigated SpaA protein from type 1 Fujisawa strain corresponding to the above SpaA protein and a gene encoding said protein to reveal that SpaA protein from type 1 Fujisawa strain is one with a molecular weight 69 kDa that has a sequence of 626 amino acid residues with one more, i.e. nine, homologous sequences of repeat at its C-terminal, as compared to the type 2 SpaA protein, with the 9th repeat consisting of 19 amino acids. They demonstrated that a fusion protein of a full-length SpaA, SpaA with deletion of the homologous sequences of repeat at the C-terminal, or SpaA with deletion of a portion of the N-terminal and the homologous sequences of repeat at the C-terminal, with a histidine hexamer, exhibited a protective effect to infection (see e.g. Non-patent references 3 and 4).
Watanabe et al. also reported that a polypeptide of 46.5 kDa prepared by deleting the homologous sequences of repeat at the C-terminal and a secretion signal sequence at the N-terminal from Erysipelothrix rhusiopathiae SpaA protein could be a protective antigen to infection (46.5 kDa protective antigen; named “46.5 KPA”)(see e.g. Patent reference 1).
On the other hand, promotion of productivity of a candidate protein for vaccine has been attempted. For instance, there is a report that 46.5 KPA could successfully be expressed for secretion out of the cells using Brevibacillus choshinensis as a host cell (see e.g. Patent reference 2). With this expression system, about 50% of an expressed protein becomes insoluble due to coagulation in culture. According to the report, purification of said insolubilized 46.5 KPA was performed by filtering a culture with ultrafiltration membrane, suspending the insoluble materials recovered on the membrane in an alkaline solution, and recovering the solubilized 46.5 KPA. Thus, this purification process requires at least three steps: (1) condensation through ultrafiltration under neutral to weak alkaline condition (pH 7 to 9.5); (2) recovery of a filtration fraction through ultrafiltration under strong alkaline condition (pH 10.0 to 12.0); and (3) purification of the ultrafiltration fraction by ion exchange chromatography.
When SpaA gene is expressed in E. coli, most of the protein may be expressed as a soluble protein and hence the purification process for insoluble materials as described above may not be applied. A culture may contain, other than SpaA protein of interest, various contaminants such as cell debris of E. coli, components from a culture medium, metabolic products produced while culture, etc. It is not easy to efficiently recover and purify the soluble SpaA protein of interest from such admixtures of contaminants. In general, a vaccine for animals, unlike a vaccine for human, would not be accepted by a stockbreeder unless it is low priced as well as in high purity and high quality. Accordingly, a manufacturer of a vaccine for animals is always required for improvement in a process for production and a process for recovery and purification that enables treatment in large scale and reduction of cost for production.    Patent reference 1: Japanese patent publication No. 2000-279179    Patent reference 2: Japanese patent publication No. 2002-34568    Non-patent reference 1: Garan, J. E. et al., (1990) Infect. Immun., 58. p. 3116-3121    Non-patent reference 2: Makino, S. et al., (1998) Microb. Pathog. 25, p. 101-109    Non-patent reference 3: Imada, Y. et al. (1999) Proc. Jpn. Pig. Vet. Soc. 34, p. 12-    Non-patent reference 4: Imada, Y. et al. (1999) Infect. Immun. 67 (9), p. 4376-4382